CHARACTERIZING TEREBRIDAE TOXINS

拟肢虫毒素的特征

• 批准号: 8169189
• 负责人: Mande N. Holford
• 金额: $ 0.35万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169189
• 关键词: Acetonitriles; Computer Retrieval of Information on Scientific Projects Database; Data; Detection; Exhibits; Funding; Grant; High Pressure Liquid Chromatography; Institution; MALDI-TOF Mass Spectrometry; Measures; Methanol; Organic solvent product; Peptides; Protocols documentation; Research; Research Personnel; Resources; Solutions; Source; Taiwan; Tarantula Venoms; Time; Toxin; United States National Institutes of Health; Venoms; Work; absorption; aqueous; disulfide bond; method development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The peptides present in the crude venom of Terebridae gutatta, argyosia, maunti and formosa were measured by LC-MS on the Orbitrap. Only the gutatta and argyosia venom had enough material to allow off-line separation. These two venoms were separated by RP-HPLC and the fractions exhibiting strong UV absorption at 214 nm were analyzed with MALDI TOF mass spectrometry to determine the accurate mass and number of disulfide bonds of potential toxins in these fractions. The Terebridae aergyosia venom showed hardly any material using UV detection, but Terebridae gutatta showed 5 major peaks. Unfortunately using a standard reduction protocol all of the gutatta peptides precipitated. The reduction protocol was optimized by increasing the amount of organic solvent (acetonitrile and methanol). Using high organic solvent the toxins remained in solution upon reduction. Toxins with masses ranging from 3700 to 5030 Da were identified. The majority of toxins (5) contained 6 disulfide bonds and one toxin contained 8 disulfide bonds. To date, we are not able to obtain fragmentation data of these toxins as we have not yet determined conditions that keep the toxins in solution and allow analysis with RP HPLC. We are currently working out protocols that keep these hydrophobic toxins in solution and at the same time retained on RP-HPLC columns. This method development is also necessary for analyzing tarantula venoms that also precipitate out of aqueous solution upon reduction.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 在 Orbitrap 上通过 LC-MS 测量了 Terebridae gutatta、argyosia、maunti 和 formosa 的粗毒液中存在的肽。只有古塔塔毒液和银蛇毒液有足够的材料进行离线分离。 通过 RP-HPLC 分离这两种毒液，并使用 MALDI TOF 质谱分析在 214 nm 处表现出强 UV 吸收的级分，以确定这些级分中潜在毒素的准确质量和二硫键数量。使用紫外线检测，Terebridae aergyosia 毒液几乎没有显示任何物质，但 Terebridae gutatta 显示了 5 个主要峰。不幸的是，使用标准还原方案，所有古塔塔肽都沉淀出来。通过增加有机溶剂（乙腈和甲醇）的量来优化还原方案。使用高有机溶剂，还原后毒素仍保留在溶液中。鉴定出质量范围为 3700 至 5030 Da 的毒素。大多数毒素 (5) 含有 6 个二硫键，一种毒素含有 8 个二硫键。迄今为止，我们无法获得这些毒素的碎片数据，因为我们尚未确定将毒素保持在溶液中并允许使用反相高效液相色谱进行分析的条件。我们目前正在制定方案，将这些疏水性毒素保留在溶液中，同时保留在 RP-HPLC 柱上。这种方法的开发对于分析狼蛛毒液也是必要的，狼蛛毒液在还原时也会从水溶液中沉淀出来。